MALDI and LDI are methods of producing ions from sample material. The term “MALDI” refers to “matrix assisted laser desorption/ionization”. The term “LDI” refers to “laser desorption/ionization”. The most common way of detecting the ions produced by these ion sources is by mass spectrometry.[1-3] Thus the ion sources (MALDI and LDI) are commonly integrated with a mass spectrometer (MS). The most common type of mass spectrometer used in this application is a time of flight (TOF) mass spectrometer. Such an ion generation and detection process is therefore sometimes referred to as MALDI-TOF-MS. This process is described in detail in the Applicant's prior international application No. PCT/CA01/01496 filed 23 Oct. 2001 and entitled “Method and Apparatus for Producing a Discrete Particle” (WO 02/035553 A3), the disclosure of which is incorporated herein by reference.
The type of laser most commonly used in MALDI and LDI applications is a N2 laser. The pulsed output of this laser is focused to a spot size on the order of approximately 200 μm in diameter. MALDI produces ions in discrete events, sometimes termed ion packets, because a pulsed laser is used in the MALDI source. Though any mass spectrometer can be used to detect the ions in an ion packet, a TOF-MS detector is best suited to resolving packets of ions as opposed to a continuous stream of ions. The OF instrument accepts a packet of ions and separates those ions based on differences in their masses, which is related to ion velocity differences by K.E.=0.5 mv2. In a constant DC field, the acceleration of all ions in a population will impart the same kinetic energy into all ions, and thus, because K.E.=0.5 mv2, a lighter ion will have a higher velocity than a heavier ion. The ions drift, and separate according to their velocities in a field-free tube. The arrival time of the ions at the end of the tube is recorded, and that time is related to the m/z of each ion.
The MALDI source is sometimes referred to in the mass spectrometry literature as a “soft” ionization source. The term “soft” implies that this ion source allows for the detection of intact compounds, even though the compounds are considered fragile (i.e. the compounds easily decompose with the addition of energy). An example of a common MALDI-TOF-MS application is the detection of peptides generated by proteolytic digestion of proteins in a sample, or proteins, oligossacharrides, RNA, DNA and other polymeric materials.[4-7] The MALDI technique may also be effective in analyzing other large biomolecules. One reason that MALDI has become a very successful and widely used technique for preparing gas-phase ions of biomolecules for mass spectrometry is that the preparation of discrete crystallized sample spots is amenable to high-throughput automated analyses.
The MALDI ion source involves the irradiation of a sample using a pulsed laser that causes the desorption/ionization of molecules in the sample spot.[8] Irradiated samples can be in a solid or liquid form, though solid samples are more commonly encountered. A solid sample is prepared by mixing an aliquot of the sample with an aliquot of a matrix solution, then the mixture is delivered (i.e. pipetted) onto a substrate and volatile solvents are allowed to evaporate, leaving behind a solid residue that contains the non-volatile species from the sample plus matrix compound(s). It is believed that the MALDI source results in little fragmentation of the analyte compounds because the technique involves the use of a matrix that is mixed with the sample at a mole ratio of ˜1000:1 chromophore:analyte. The matrix is in fact a chromophore that absorbs the output of the pulsed laser used in the MALDI experiment. The matrix absorbs the radiation from the pulsed laser and is itself vaporized and partially decomposed. During the vaporization, analyte molecules are also carried into the gas phase and by either direct ionization or secondary ionization, the analyte molecules become ionized.[9, 10] Direct ionization is the absorption of the laser radiation and ejection of an electron from the analyte. Secondary ionization refers to gas-phase ion-molecule reactions in the plume of material desorbed by the laser. The extent of secondary ionization is not well characterized in the prior art.
The ease with which an analyst prepares a sample for characterization by MALDI is itself easy, simple, and fast: an analyst need only mix the sample with a matrix solution. An aliquot, or all of that mixture is then deposited onto a substrate and the volatile solvent in that mixture is allowed to evaporate dry to leave a dry, solid residue. That residue is then targeted with the laser in the MALDI-TOF-MS instrument. In principle, the preparation of sample material for MALDI-TOF-MS analysis is trivial. In reality, the most frequently encountered problem in the technique is that the sample is simply not detected. There are many reasons for that, such as the threshold level for laser power prior to observing analyte ions. Because of this and other easily and commonly observed characteristics of MALDI, it is widely believed that the detection of an analyte compound in a MALDI experiment critically depends on the crystallization of the analyte compounds with the matrix.
The Applicant's prior international application referred to above (WO 02/035553 A3) describes electrodynamically levitating a sample particle, which may include a solid member, a droplet, a single molecule, or a cluster of molecules, and delivering the particle to a target location. This process is sometimes referred to as “wall-less sample preparation” (WaSP). Briefly, in one embodiment the WASP technology involves the use of an ink-jet droplet generator to create droplets from a starting solution. In order to levitate the droplets in the electrodynamic balance (EDB) a net charge is induced on to the droplet. Though other forms of levitation could be used, each would have their own constraints on the physical and chemical composition of the droplet. The volatile solvents in the starting solution, such as methanol and water, rapidly evaporate (i.e. typically within 1-2 seconds) from the droplet. The evaporation of volatile solvents concentrates the non-volatile (plus low volatility) solutes that were in the starting solution inside what is now descriptively referred to as the levitated droplet residue. That droplet residue is then deposited onto a target substrate. Translating the substrate relative to the EDB, or vice versa, and repeating the process of creating and levitating a droplet followed by the deposition of that residue allows a user of WaSP to pattern multiple spots of materials onto a substrate.
As described herein, smaller spots of sample may prepared by adjustment of the time invariant (i.e. DC) and time variant (i.e. AC) electrical potentials applied to the EDB. Since the EDB is in effect an atmospheric pressure or “Paul trap”, one can describe droplet/particle stable levitation in a-q space. The term “Paul trap” is in reference to the contributions made by Wolfgang Paul which led to his being awarded a (Physics) Nobel prize.[11]
Alternatively, a user of WASP could employ the use of droplets that are themselves not stable. Specifically, it is possible to allow the levitated droplet to become electrically unstable by adjustment of the starting solution composition, induction potential, or the environmental conditions within the levitation chamber. If a droplet with net charge becomes unstable, it can undergo one or more Coulomb explosion events.[12] If so, the material ejected from the droplet can be directed onto different regions of a target substrate by introducing an electric field orthogonal to the direction for deposition of the levitated droplet onto a substrate.
Moskovets et al. have shown that by placing two discrete sample traces within 100 μm to 4 mm of one another, and performing MALDI on them separately, mass detection accuracy gains were achieved for traces in the center and edge of their MALDI target plate [13]. Moskovets et al. do not teach the advantages of irradiating two or more closely spaced samples simultaneously.
Other sample preparation methodologies are known in the prior art. The Karger group has developed, and patented, the use of a vacuum deposition of the liquid emerging from a capillary electrophoresis (CE) column.[14, 15] The purpose of the vacuum is to remove volatile solvent quickly to reduce the extent to which sample smears on the plate. The utility of this technology has been demonstrated by the Karger group to enable the deposition of materials as tracks (i.e. eluant from a separation) alongside another track of material, such as an internal standard. In their work, the substrate was moved underneath the laser spot (i.e. rastered), effectively sampling both tracks of materials serially, but not simultaneously, between the two tracks of materials. The objective of their work described in this manuscript was on improving mass accuracy of the compounds detected in a MALDI-TOF-MS experiment.
The Li group has developed a nanoliter sample preparation platform to the extent that they now refer to the technology as a nanoliter chemistry station.[16, 17]
Smith has described the use of open channel electrophoresis for coupling separations to MALDI-TOF-MS.[18] Along the same lines, a microfluidic sample preparation system has also been developed for preparing sample material for MALDI-TOF-MS.[19] Several other groups are preparing spots of sample materials using droplet dispensers with direct deposition onto a substrate.[20-24]
Early applications of MALDI time of flight mass spectrometry (TOFMS) in the study of biomolecules using an organic matrix used sample spot sizes (˜5 mm2) that were much larger than the laser spot diameter (˜10−3 mm2) used for analysis. [25-27] Since then, this approach has been almost universally accepted because, by irradiating a single sample spot that was larger than the laser diameter, one could ensure that only the sample of interest was being probed and sample preparation using micropipettes was trivial. [28-32] However, as experienced by most practitioners of MALDI, even though only a single sample spot is analyzed the large sample spot does not produce uniform signals over its entire area because of microheterogeneity in the sample.[33,34] This has led to the notion of ‘hot’ or ‘sweet’ spots, small regions within a large sample spot from which large fluxes of analyte ions are detected. One of the responses to this problem has been to devise new methods that create smaller sample spots that reduce the need to search for hot spots because almost the entire sample is irradiated.[35-41] These approaches have also fulfilled demands for less sample consumption and the preparation of higher densities of sample spots on a single MALDI plate to increase throughput. For example, 100×100 to 400×400 μm microfabricated silicon picoliter vials that hold sample dispensed through a piezoelectric droplet generator have been shown to increase sensitivities 10-50 times relative to dried-droplet preparation. [39] Note that the laser spot diameter used to analyze the samples deposited in the picoliter vials was ˜100 μm in diameter and thus it and the sample spots were nearly the same size. Similarly, the development of a microspot sample preparation technique using a picoliter syringe demonstrated that as little as 20 pL of sample volume could be manipulated to create sample spots of ˜100 μm in diameter, enabling the analysis of the contents of a single red blood cell (˜87 fL volume).[42] The laser spot size that was used for analysis was in the shape of an oval, 50×180 μm, so again the sample spot and laser spot size were nearly equal.
Another advantage of small sample spot sizes, high sensitivity, was clearly demonstrated by the detection of 42 zeptomoles (25 000 molecules) of substance P from a 0.08 mm diameter microspot.[43] That study also revealed that the minimum absolute detection limit was set by the number of molecules per μm2 (>5 molecules). This suggests that if the sample spot size can be decreased while maintaining the required analyte density, very high density sample spot arrays can be produced on MALDI plates for high-throughput analyses at high sensitivities while consuming very small volumes of sample. Recent applications of high-density sample spot preparations include the coupling of separations techniques such as liquid chromatography and capillary electrophoresis to MALDI for analysis by offline mass spectrometry[28,44-46] and the spotting of matrix onto a sample for subsequent imaging of the ions on the surface by mass spectrometry.[47-50] As with many other disciplines, there are clear advantages for MALDI sample preparation to move towards smaller and smaller dimensions.
While other sample preparations methodologies useful for MALDI-TOF-MS are known in the prior art, improved methods for controllably depositing microspots of sample material on a target substrate in close proximity to one another are desirable as are improved methods for simultaneously irradiating adjacent microspots.